The application of recombinant DNA technology to industrially important organisms such as Streptomyces and related actinomycete genera requires efficient gene-cloning and transformation procedures. Polyethylene glycol (PEG) induced plasmid transformation of protoplasts has allowed for the development of gene cloning procedures for several species of Streptomyces and related genera. Thus, Acebal et al, 1986, FEMS Microbiol. Lett. 35: 79-82, describe a method for transforming S. wadayamensis, a .beta.-lactam antibiotic producer; Bibb et al., 1978, Nature 274: 398-400 describe high frequency transformation of plasmid DNA into Streptomyces; Lampel and Strohl, 1986, Appl. Environ. Microbiol. 51: 126-131, describe transformation and transfection of anthracycline-producing streptomycetes; Matsushima and Baltz, 1985, J. Bacteriol. 163: 180-185, describe efficient plasmid transformation of S. ambofaciens and S. fradiae; Yamamoto et al., 1986 J. Antibiot. 39: 1304-1313 describe transformation of S. erythraeus; Pidcock et al., 1985, Appl. Environ. Microbiol. 50: 693-695, describe transformation of Thermomonospora fusca protoplasts; and Matsushima et al., 1987, J. Bacteriol. 169:2298- 2300, describe transformation of Amycolatopsis (Nocardia) orientalis. Thus, transformation of various species and strains of Streptomyces and other actinomycetes has been achieved, but in many cases at relatively low frequencies of transformation. Low frequencies of transformation may be due to a multiplicity of reasons, including inefficient uptake of DNA, restriction enzyme digestion of DNA after uptake or difficulties in preparing and/or regenerating protoplasts.
Several difficulties associated with the above-referenced protoplast transformation methods using double-stranded plasmid DNA has, however, impeded the wide application of recombinant DNA technology in many species of Streptomyces and related genera. Most streptomycetes produce a multiplicity of restriction enzymes (see Cox and Baltz, 1984, J. Bacteriol. 159: 499-504; Lomovskaya et al., 1980, Microbiol. Rev. 44: 206-229; McHenney and Baltz, 1988, J. Bacteriol. 170:2276-2282; and Engel, 1987, Appl. and Environ. Microbiol. 53:1-3) that can dramatically decrease the efficiency of plasmid transformation and phage infection. See Matsushima and Baltz, 1985, J. Bacteriol. 163: 180-185; Chater and Wilde, 1980, J. Gen. Microbiol. 116: 323-334; Chater and Wilde, 1976, J. Bacteriol. 128: 644-688; and Chater and Carter, 1978, J. Gen. Microbiol. 109: 181-185. The problem caused by restriction enzymes is often compounded by the rigid procedural requirements for efficient uptake of plasmid DNA and protoplast regeneration. Physiological conditions for cell growth that might minimize the expression of restriction enzymes often inhibit efficient uptake of DNA, plasmid replication and protoplast regeneration.
Several approaches attempting to solve these difficulties have been described. One approach is the use of a phage transduction system for gene cloning and transfer to partially overcome restriction as described in U.S. patent application Ser. No. 07/020,807 (attorney docket no. X-7088) filed Mar. 2, 1987. In a transduction system, the transducing DNA can be packaged into phage particles, which can attach and inject DNA, and thus transduce intact cells, avoiding the use of protoplasts. Intact cells may tolerate a broader range of culture conditions, especially temperature of incubation, better than protoplasts. Since many host restriction systems become less active as the temperature of incubation varies from the temperature of optimal growth, a transduction system using intact cells and varying the temperature of incubation may help to reduce the effectiveness of host restriction enzymes. In addition, raising the multiplicity of infection (m.o.i.), and thus increasing the amount of transducing DNA introduced into a cell, may be used in an attempt to overwhelm host restriction systems. However, a limitation of this system is that only those host cells which are susceptible to infection by such phage may be effectively transduced. In the transduction system of U.S. patent application Ser. No. 07/020,807 a segment of bacterophage FP43 DNA, designated hft for high frequency transduction, was cloned into plasmid pIJ702. The resulting plasmid pRHB101 could be efficiently packaged into FP43 phage heads as linear concatemers to form effective transducing particles.
Another approach to the problem of restriction that was used by Matsushima et al., 1987, Mol. Gen. Genet. 206:393-400 is the development of transformable mutants of Streptomyces fradiae defective in several restriction systems. Streptomyces fradiae is an important industrial microorganism used to produce the macrolide antibiotic tylosin. In the process of developing S. fradiae as a host for gene cloning, Cox and Baltz, 1984, J. Bacteriol. 159:499-504, and Matsushima and Baltz, 1985, J. Bacteriol. 163:180-185, observed that S. fradiae expresses potent restriction systems for bacteriophage DNA and plasmid DNA, respectively. Other industrially important species of Streptomyces have been found to be similarly highly restricting. Therefore, to efficiently clone DNA from heterologous sources into such a highly restricting strain as S. fradiae, mutants were developed by Matsushima and Baltz, 1987, Mol. Gen. Genet. 206:313-400, lacking one or more restriction enzyme systems. A major problem with this approach is that such mutants must be developed on a strain-by-strain basis and the development and selection of such mutants is not trivial and very time consuming, requiring multiple selection steps. For example, Matsushima et al., 1987, Mol. Gen. Genet. 206: 392-400, describe four initial rounds of mutagenesis accounting for four discrete increases in transformation efficiency with the loss of one modification system, a fifth round of mutagenesis causing the loss of three modification (and presumably restriction) systems, and a final round of mutagenesis causing a large increase in transformation efficiency. This suggests that wild-type S. fradiae strains may express greater than five functional restriction systems.
In one aspect, the present invention comprises novel single-stranded DNA vectors and methods of transformation with such vectors that may act to bypass host cell restriction systems and thus permit the efficient transfer of DNA between various species of Streptomyces and between strains of E. coli and Streptomyces. These vectors may also be packaged into phage particles, and fused with the cell membrane to permit DNA transfer. These cloning vectors are bifunctional, containing both a Streptomyces origin of replication (for example, SCP2.sup.* -ori or pIJ101-ori) and an E. coli origin of replication. The present invention thus allows efficient gene transfer between many species of Streptomyces or several other genera of actinomycetes, and E. coli.
A number of single-stranded DNA vectors have been described. These vectors are summarized in Table 1.
TABLE 1 ______________________________________ Vectors Reference or Commercial Source ______________________________________ pEMBL series Dente, et al., 1983, Nucl. Acids Res. 11:1645-55. pBluescript, Stratagene, 11099 North Torrey Pines pBluescribe Road, La Jolla, CA 92037 fBB101, fBB103 Barany, 1982, Microbiology 51:125- 129. pTZ18, pTZ19 Mead et al., 1986, Protein Engineering 1:67-74; also available as pTZ18R and pTZ19R from Pharmacia, Molecular Biology Division, Piscataway, N.J. 08854 pKUN9, pKUN19 Konings et al., 1987, Methods in Enzymology 153:12-34; European Patent Application 86201252.3 pGBT518, Gold Biotechnology, 5050 Oakland pGBT519, pGBTT13 Avenue, St. Louis, Missouri 63110 pYK331, pYK332, European Patent Application pYK333, pYK335, 84112724.4 pYK336 ______________________________________
The single-stranded vectors listed in Table 1 were constructed for the following uses: cloning and dideoxy DNA sequencing (Sanger et al., 1980, J. Mol. Biol. 143: 161-178); site-directed mutagenesis (Zoller and Smith, 1982, Nucl. Acids Res. 10:6487-6500); S1-mapping (Ciliberto et al., 1983 Gene 2:95-113); mRNA cloning (Heidecker and Messing, 1983, Nucl. Acids Res. 11:4891-4906); expression of cloned DNA in E. coli (Slocombe et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 5455-5459); production of single-stranded hybridization probes (Hu and Messing, 1982, Gene 17;271-277); heteroduplex analysis and in vitro transcription RNA (Mead et al., 1986, Protein Engineering 1:67-74). None of the single-stranded vectors of Table 1 have been used or suggested for use in transformation of Streptomyces, other actinomycetes, and/or E. coli. The fBB101 and fBB103 vectors described by Barany, 1982, Microbiology 51: 125-129, were used in transformation experiments with Streptococcus pneumoniae. However, when the transformation efficiencies of single-stranded DNA and double-stranded DNA of the fBB101 or fBB103 vectors were compared, the single-stranded DNA transformed Streptococcus pneumoniae at efficiencies 50-100 fold lower than those obtained using double-stranded DNA. Thus, the results of Barany with fBB101 and fBB103 suggest that a single-stranded DNA vector may not be as useful as a double-stranded DNA vector in transformation.
In contrast to these previously described single-stranded vectors and their uses, the vectors of the present invention represent novel hybrids among a Streptomyces plasmid vector, an E. coli plasmid vector, and an E. coli bacteriophage. These vectors are bifunctional for Streptomyces and E. coli because they contain both a Streptomyces and E. coli origin of replication. Finally, the single-stranded vectors of the present invention are useful in a method of transformation because they appear to bypass Streptomyces host cell restriction systems and thus permit the efficient transfer of DNA into highly restricting strains of Streptomyces and other organisms that are not able to be transformed at useful frequencies by other methods. These single-stranded vectors are able to transform Streptomyces at frequencies higher than, or at least comparable to, those frequencies obtained using double-stranded DNA.